Regulation of vacuolar V-ATPase activity and Dynamic control of vacuolar functions

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Y. Anraku et al. found a novel ATPase on the vacuolar membrane of Saccharomyces cerevisiae in 1980 It functions as a major proton pump of the vacuole. The enzyme is a supra-macromolecular complex consisting of at least 13 subunits. Its physiological functions are proposed to participate in dynamic controls of vacuolar functions, particularly the regulation of intracellular calcium ion and proton concentrations. In this project, we intended to study a comprehensive view on the regulation of the V-ATPase activity and vacuolar dynamics in cell physiology.The results are : 1) Applying a newly established method of whole vacuolar patch clamp. we determined an ATP-dependent inward current of 100 pA across the vacuolar membrane with a proton/ATP ratio of 3.5. This provides direct evidence that the enzyme acts as a proton pump under physiological conditions 2) The structure and function of novel genes, VMA14 and VMA15 were determined. The products have a regulatory role for the assembly of subunits of the V-ATPase on the vacuolar membrane. 3) A novel gene, VIS1 was isolated and its structure was determined. The product (214 amino acid residues) is a membrane protein enbedded in the vacuole. Its function relates to a regulatory system for Ca^<2+>-sensitive growth with a cross interaction of the UBP signaling network.